Abstract The broad long-range goals of this project are to understand how two major classes of chemotherapeutic agents cause DNA damage and how cells deal with DNA-protein crosslinks which are induced by these agents. The repair of DNA-protein crosslinks is very poorly understood, in spite of their prevalence from many chemotherapeutic agents as well as radiation and other chemicals. The quinolone antibiotics, which target bacterial DNA gyrase, stabilize a reaction intermediate in which the enzyme is covalently attached to a broken DNA molecule via phosphotyrosine bonds. Anticancer drug 5-azacytidine, on the other hand, leads to covalent complexes between cytosine methyltransferase and DNA at the recognition sites of the enzyme, with no DNA break in the complex. We have shown that both classes of inhibitors lead to blockage of the replication fork at sites where the enzymes are covalently bound to DNA. The first aim seeks to elucidate the mechanism of DNA breakage and cytotoxicity after treatment with quinolones, with an emphasis on the role of several genes including dnaQ, recQ, xseAB, and ruvAB. The second aim analyzes mutants that are hypersensitive to 5-azacytidine, which induces covalent methyltransferase-DNA complexes. The basis of their hypersensitivity will be probed with a number of experimental tests, and a major goal is to identify a subset of the mutants affected for repair of DNA damage from the DNA-protein crosslinks. The third aim uses biochemical approaches to investigate the repair and processing of DNA-protein crosslinks formed by both classes of chemotherapeutic agents. This includes biochemical analyses of intermediates formed in vivo, as well as in vitro experiments where we analyze the fate of DNA-protein crosslinks in reactions with cell extracts or purified proteins. Finally, in the fourth aim, we follow up on our recent observation that the tmRNA translational quality control system is important for survival after 5-azacytidine treatment. We have proposed that the DNA-protein crosslinks block transcription, which leads to blockage of the coupled translating ribosomes, to trigger the tmRNA system into action. Aspects of this model will be tested, including the role of the site- specific crosslinks in triggering the tmRNA system and the fate of the RNA and RNA polymerase in the blocked complexes.